Ion channels that mediate Na reabsorption by tight epithelia will be characterized, and the mechanisms by which these channels are regulated will be investigated. The major model tissue to be used will be the cortical collecting tubule of rat kidney. Apical membrane channels which are selective for Na ions and sensitive to the diuretic amiloride will be studied using the patch-clamp technique to identify ionic currents through individual channels. The biophysical nature of theses channels will be explored by assessing the selectivity of the channels for various cations, the conductance of the channels at different concentrations of permeant ions, the rates of spontaneous channel opening and closing, and the details of the ability of K-sparing diuretics to inhibit channels function. The regulation of the channels by various factors including hormones (aldosterone and ADH), external and internal ion concentrations (Ca, H and Na), the metabolic state of the tissue, and the activities of enzymes which methylate and phosphorylate membrane proteins will also be investigated. Finally, group-specific reagents will be employed to discover the chemical nature of important amino acids which govern the conductance and regulation of the channels. This project will increase the understanding of the importance process of NaCl reabsorption by epithelia, defects in which have been implicated in such diseases as cystic fibrosis, congestive heart failure and diarrhea.